The goal of this research is to dramatically improve the ability to clone and analyze large and unstable DNA fragments. We propose to develop a novel linear cloning vector to maximize stability of cloned DNA in the bacterial host. The vector will use the replication proteins of the phage Phi29 to achieve the highest accuracy of replication for all DNA inserts, including AT-rich, repetitive, or structurally unstable genes. The cloning method will be quick and easy for DNA of any size range (0-100 kb), only requiring ligation of small vector arms to the DNA, followed by transformation of bacteria. This system will surpass existing standards for stability, fidelity, and lack of bias, exceeding the limits of our previously developed pJAZZ linear cloning vector. Importantly, it will enable straightforward cloning of fosmid- or BAC-sized DNAs. The vector will have minimal bacterial sequences, providing significant benefits for mammalian cell transfection, therapeutic protein production, stem cell research, and ultimately gene therapy. The cloned DNAs will be bound to nuclear localization signals for enhanced delivery and expression in mammalian cells. This Phi29 vector is urgently needed to help discover treatments for human illness. For example, the genome of the human malaria parasite, Plasmodium falciparum, is among the most difficult genomes to clone, due to its repetitive, highly AT-rich nature. We will collaborate with the Wellcome Trust Sanger Institute to construct and sequence long-insert genomic libraries of Plasmodium falciparum. This unique resource is critically needed to enable genetic research on this deadly pathogen.